Rescuing plasmid from dead E.coli stock.
Frank O. Fackelmayer
fof1 at chclu.chemie.uni-konstanz.de
Fri Dec 6 05:04:00 EST 1996
<Pine.SGI.3.91.961205095147.17816B-100000 at hancock.microbiology.ubc.ca>,
emma at HANCOCK.MICROBIOLOGY.UBC.CA (Emma Macfarlane) wrote:
> I remember reading some time ago on this newsgroup about retreiving
> plasmid from dead plates to re-transform into E.coli, but I can't
> find reference to it in the archives. Is it possible to
> do the same thing with frozen stocks? We have an important construct
> in a sickly E.coli strain that is now refusing to grow. I tried
> taking a scraping of cells, boiling it up in water, spinning it down
> and using the supernatant to transform DH5a. This didn't work.
> If anyone has an alternative suggestion I would appreciate hearing it.
> Please reply direct to my email address emma at hancock.microbiology.ubc.ca
> as I am not a regular subscriber to the newsgroup.
> Thanks in advance
I recommend you do a normal miniprep (Birnboim) of a small aliquot of your
old stock and use some of the DNA for a new transformation. I have done
that successfully several times, with good success. Normally I take 1ul of
50ul from the miniprep for a transformation (dont run it on a gel, you
wont see anything as the yield is very low). This is usually enough to get
a lot of clones, even when you start from only 10ul of glycerol stock. You
can even do the recovery with dried-out agar plates: cut out a piece where
a clone was, drop the piece in GET for several hours to overnight, then do
a normal miniprep without removing the agar pieces before (looks terrible,
but works). Of course you can also use stab culture stocks to do the
Hope it helps,
Dr. Frank O. Fackelmayer
Division of Biology
University of Konstanz
More information about the Methods